It is now clear that GPCR signaling is pluridimensional. Defining mechanisms for receptor signaling pathways is essential for understanding many aspects of cell biology, as well as for effectively targeting signaling pathways for drug discovery. Ligands of receptors do not activate all pathways equally but rather can exhibit a bias towards some pathways at the expense of others. Signaling pathway-specific ligands or biased ligands that selectively activate one pathway over another, as well as behave as agonists in one pathway but antagonists in another pathway have greatly impacted our understanding of intricate GPCR signaling pathways and GPCR drug discovery. In addition to G-protein and ?-arrestin signaling pathways, signal adaptor protein 14-3-3 is another cellular effectors activated by GPCRs. The first evidence of 14-3-3 as a cellular effector of GPCRs was demonstrated with the ?2-adrenergic receptors (?2ARs). Interaction of 14-3-3 and ?2ARs is ligand-dependent. 14-3-3 proteins are ubiquitously expressed in cells, but their highest expression is found in the brain. 14-3-3 proteins have been implicated in a number of neurological disorders such as Alzheimer's disease, Parkinson's disease, schizophrenia, bipolar disorder based on evidence from both clinical and laboratory studies. Similar to ?-arrestins,14-3-3 proteins have no intrinsic enzymatic activity, but bring two or more proteins together to facilitate signal transduction processes. A very recent bioinformatic analysis predicts 68% neurotransmitter GPCRs including GRMs have 14-3-3 binding motifs. Interestingly, GRMs, family C GPCRs, do not recruit ?-arrestins like many GPCRs do when activated. Do they utilize 14-3-3 for additional signaling in addition to G-proteins? Lack of a user-friendly and scalable too for assessing GPCR-mediated 14-3-3 signaling could be a major reason for the signaling pathway unexploited. We plan to apply LinkLight technology to develop GPCR mediated 14-3-3 signaling pathway assays. We plan to use ADRB2 as a model to demonstrate the assay feasibility. The assay utilizes ADRB2 and 14-3-3 interaction or ADRB2/14-3-3 signal complex formation as the signal readout. Our preliminary data (transient expression and stable expression experiments) showed ADRB2/14-3-3 interaction or signal complex formation is agonist concentration-dependent. We also compared ADRB2/14-3-3? and ADRB2/?-arrestin-2 LinkLIght assays in response to various agonists, partial agonists, and antagonists. The preliminary data showed that the overall patterns of various ligand responses were similar, but there were some differences in relative signal strength and potency (EC50). Interestingly, ADRB2 antagonists (based on G-protein signaling) showed a partial activity in ADRB2/?-arrestin assay, but had no activity in ADRB2/14-3-3 assay. Our preliminary results showed that ligands are not created equal based on different signaling pathways. Based on the observation, it prompts us to profile existing drugs for their ?-arrestin and 14-3-3 signaling pathways. Historically, GPCR drug discoveries rely on G-protein signaling pathways to assess compound activity such as many old antipsychotic drugs. Their activity on ?-arrestin and 14-3-3 signaling i unknown. Although having demonstrated therapeutic benefits, these drugs also have serious side effects. Despite huge efforts spent by the pharmaceutical industry, the options for developing safer and more efficacious antipsychotic drugs remain elusive. Could therapeutic and side effects are due to specific signaling pathways? I plan to use D2R, an important antipsychotic drug target as a model to investigate a panel of D2R ligands for their activity on ? arrestin and 14-3-3 signaling. It is now known that ligands do not activate all pathways equally but rather can exhibit a bias towards some pathways at the expense of others. Ligands could have 14-3-3 signaling in addition to G-protein and ?-arrestin signaling. Biased ligands could have differential activities on these signaling pathways. We plan to take the advantage of multiplex ability of the LinkLight technology to establish a dual-signaling pathway assay for assessing ?-arrestin and 14-3-3 signaling simultaneously. We plan 4 tasks for the proposal. Task1, Using ADRB2 as a model to assess GPCR-mediated 14-3-3 signaling and generating stable 14-3-3-pLuc reporter cell lines. Task 2, Assess the general applicability of GPCR/14-3-3 signaling assays with brain-derived GPCRs. Task 3, Profile and compare D2R-mediated 14-3-3 and ?-arrestin signaling pathways with known D2R ligands. Task 4, Develop GPCR/14-3-3 and GPCR/?-arrestin dual- signaling pathway assays. The assay cell lines (tools) developed in the proposal will be commercially available. We also plan to use the tools for commercial compound screening and profiling services. A CDA and MTA have signed with a major US research reagent company for potential business opportunity collaboration.